The purpose of this shared equipment grant is to obtain instrumentation to monitor fluorescent macromolecules in living cells and organelles. Our objective is to visualize and to quantitate fluorescent molecules using video intensification microscopy (VIM) and flow microfluorometry (FMF). The individual proposals reflect quite diverse interests, but share a common need for these instruments. In each proposal we have attempted to develop the scientific problem then indicate how this instrumentation will play a key role in its solution. In most instances the information being sought, for example, the effect of an introduced actin crosslinking protein on the shape of a living cell or the dynamics of the uptake and degradation of lipoproteins by living fibroblasts, simply cannot be obtained in any other way. The instrumentation requested has three basic functions which the individual projects utilize to varying degrees. The FMF measures the global signal from single cells stained with fluorescent reagents, accumulates these measurements and then gives a frequency distribution of the fluorescence from a large number of cells. The FMF can also fractionate or sort specifically stained cells from the general population. The VIM, on the other hand, can provide information on the local geography of a probe within a single cell and can be used to follow changes in its distribution as a function of time. The three capabilities provide a unique approach to the measurement, selection and finally observation of macromolecules which have been introduced into or taken up by living cells. Four of our projects exploit this approach to the fullest and propose to study, in living cells, the dynamics of various macromolecules including; tubulin, actin associated proteins, CDR, low density lipoproteins and Fab fragments. Another proposal makes use of both the analytical and sorting capabilities of the FMF to characterize and fractionate chromosomes for more traditional biochemical work. The final project focuses on the cell cycle and uses the FMF to analyze rapidly the DNA frequency distributions in cells transformed with temperature sensitive (SV-40) viruses.